The complete mitochondrial genome and description of a new cryptic Brazilian species of Metopiellus Raffray (Coleoptera: Staphylinidae: Pselaphinae)

Metopiellus Raffray, 1908 is a genus of South American rove beetles typically found in tropical humid forests. Here we describe a new cryptic species from Eastern Amazon, in northern Brazil, Metopiellus crypticus Asenjo sp. nov., and its major morphologic diagnostic features, which were photographed and illustrated. In addition, we bring the complete mitochondrial genome sequence of M. crypticus sp. nov., and its position within the phylogenetic context of the family, including previously available mitogenomes of Staphylinidae species.

The aim of this study was to describe a new species from Brazil that was collected in the state of Pará, northern Brazil. The new species was found inhabiting forest areas, similar to other species in the genus, but it was also found in savanna-like environments. We

Field collection and sequencing
A total of 12 specimens of Metopiellus crypticus Asenjo sp. nov. were collected in forest areas from the Serra dos Carajás in Pará, Brazil, in which the individuals were more abundant, and only two specimens from the canga, a savanna-like environment from the region (Figs. 1B-1C), according to the sampling permit 49.994, granted by ICMBio/MMA. Various collection methods were used in all sites (hand collection, litter sampling and hay-bait traps and soil sampling by flotation), but specimens were found only in litterassociated methods (hay-bait traps and soil sampling by flotation). Therefore, it is likely that the edaphic environment is characterized as a preferred habitat for populations of Metopiellus crypticus Asenjo sp. nov. The specimens were immediately fixed in 99% ethanol within a 2 mL centrifuge tube and transported to the laboratory. Total genomic DNA was extracted from three specimens from the type population with the DNeasy Blood & Tissue kit (Qiagen), following the manufacturer's protocol for insect samples, being deposited at the DNA bank of the Instituto Tecnológico Vale (ITV) under the accession numbers ITV10661, ITV21026 and ITV21027. Paired-end libraries were constructed from ∼50 ng of genomic DNA using the QXT SureSelect kit (Agilent Technologies, Santa Clara, CA, USA), with which the DNA samples were subjected to an enzymatic random fragmentation and simultaneously bound to adapters, following the manufacturer's instructions. Then, the samples were purified and subjected to an amplification reaction using primers complementary to the adapters. Afterwards, the libraries were quantified using a Qubit 3.0 (Invitrogen, Waltham, MA, USA) fluorimeter and checked for fragment sizes in a 2100 Bioanalyzer (Agilent Technologies). Finally, the libraries were diluted in a solution of 0.1% Tris-HCl and Tween and pooled to be sequenced in an Illumina NextSeq 500 with the high-output v2 kit (300 cycles, 2 × 150 bp). Resulting raw sequencing reads with base quality <Phred 20 and length <70 bp were trimmed with AdapterRemoval v.2 (Schubert, Lindgreen & Orlando, 2016), resulting in 21,140,835 (ITV10661), 48,275,325 (ITV21026) and 49,851,478 (ITV21027) high quality pairs of reads, which were used to assemble the mitochondrial genomes using NovoPlasty 3.6 (Dierckxsens, Mardulyn & Smits, 2017). A bash script containing the commands for the cleaning and assembling steps is available as Data S1. Finally, the annotations were performed with MITOS2 (Bernt et al., 2013), with subsequent minor manual corrections using Geneious Prime 2021 (Biomatters), by comparing the annotated mitogenomes with previously available data for Pselaphinae species.

Morphological study
Specimens. The apical segments were cleared in a double boiler using 10% KOH during three minutes. Dissections were made under a Leica S8APO (16×-128×) stereo-microscope. Pictures were obtained using the AxioCam 506 color camera connected to an Axio ZoomV16 (ZEISS) stereo microscope and Photoshop CC 2021 was used for image processing, with final plates being assembled in Adobe Illustrator CC 2021. Morphological character terminology, including foveation and its abbreviation followed Chandler (2001). All measurements were made using the Leica S8APO (16×-128×) stereo-microscope, and the width/length ratios were acquired using the widest and longest parts of the respective structures, being presented in millimeters, based on the holotype. We also performed additional measurements using the paratypes, comparing with the data obtained for the holotype (Data S2).
Measurements symbols: BL body length (from margin of prolongation of head to tergite IX posterior margin) BW body width (maximum width of elytra) EL elytral length (maximum) EW elytral width (maximum) HL head length (from anterior margin of prolongation of head to head disc posterior margin) HW head width (maximum) NW neck width (minimum) PL pronotum length (maximum) PW pronotum width (maximum) In the type label data, quotation marks ('' '') separate different labels and a slash (/) separates different lines within a label. Text within square brackets [] is explanatory and is not included in the original labels.

Nomenclatural acts
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Commission on Zoological Nomenclature (ICZN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved, and the associated information viewed through any standard web browser, by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:85F589F2-AECD-4D52-B545-6363C4CE8E18 The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central and CLOCKSS.

Description
Family Staphylinidae Latreille, 1802 Subfamily Pselaphinae Latreille, 1802 Tribe Metopiasini Raffray, 1904Subtribe Metopiasina Raffray, 1904Genus Metopiellus Raffray, 1908 Metopiellus crypticus Asenjo, new species    Figure 3 Representative genetic map of the mitogenome of Metopiellus crypticus sp. nov. Disposition of all 37 mitochondrial genes. Colored arrows pointing to the left and right represent the transcription regions of protein coding genes (blue), rRNA genes (red) and tRNA genes (purple) on the L and H strands, respectively. The green and brownish bars above the arrows indicate monomorphic and polymorphic nucleotide sites among the three analyzed genomes, respectively.
Aedeagus (Figs. 2H-2J): asymmetric with paramere partially fused to form elongate plate with apex forked, the median lobe slightly bulbous at base, elongate and narrow, stronger curved laterally at apex, on edge with line of small teeth.

Mitogenome sequence and phylogenetic placement
All three assembled mitogenomes (GenBank accession numbers MZ576843 (ITV10661), MZ576844 (ITV21026) and MZ576845 (ITV21027)) presented the standard structure sequence and gene content for Metazoa, consisting of 13 PCG, 22 tRNA genes and two rRNA genes (Fig. 3). The three mitogenome assemblies ranged in size from 14,353 to 14,984 bp, with similar GC contents between 16.2% and 16.5%, and 98.3% identical sites (1.7% differences). We observed differences in nucleotide composition among the three mitogenomes of Metopiellus crypticus Asenjo sp. nov., with indel events mostly occurring in the rRNA genes (three in each locus). Also, rrnL presented 33 site substitutions as indicated by the mismatches in the alignment, one of the highest proportions of polymorphic sites within the analyzed mitogenomes (2.68% of the 1232 bp), behind only of NAD6 (3.73% of the 456 bp), excluding the tRNAs, which are considerably shorter with 63 bp on average (Table 1). Most genes were encoded in the L-strand, including nine PCGs (ATP6, APT8, COB, COX1, COX2, COX3, NAD2, NAD3 and NAD6) and 14 tRNA genes (trnA, trnD, trnE, trnG, trnI, trnK, trnL2, trnM, trnN, trnR, trnS1, trnS2, trnT and trnW). Also, ATT was the most frequent start codon, being observed in seven genes, followed by ATA in four genes, and ATG in three (Table 1). On the other hand, almost all genes presented the TAA stop codon, except for NAD3 with TAG, and the three COX genes with an incomplete stop codon (Table 1).
In the phylogenetic analysis, most of the subfamilies were recovered as monophyletic and well supported, except for Tachyporinae and Paederinae, which were polyphyletic and paraphyletic, respectively, and Staphylininae, presenting a low bootstrap support (BS = 55) (Fig. 4). Within Pselaphinae, the relationships among the sampled species were mostly unsupported (BS <70). The three specimens of Metopiellus crypticus sp. nov. grouped with maximum statistical support (BS = 100) in the longer branch within the subfamily, being recovered as sister to Batrisodes sp., although with low statistical support (BS = 42) (Fig. 4).

DISCUSSION
The new species belongs to the genus Metopiellus based on the third antennal segment being much shorter than the second (Fig. 2E); the posterior coxae contiguous or nearly so; and the mesial face of protibia being carinate and open at its base and apex (Fig. 2B) (Raffray, 1908;Park, 1942;Asenjo, Ferreira & Zampaulo Rde, 2017). One of the characters ''pronotum not being spinose'' for Metopiellus, should not be considered a good character as considerated by previous authors to define the genus since M. guanano has pronotum with four small spines (Fiorentino, Tocora & Ramirez, 2022).

Notes.
Sequenced mitogenomes based on from three different specimens, indicating the size of the transcription regions, presence of indel events, number of mismatches after the alignment of the mitogenomes, coding strand, and sequences of both start and stop codons. a For genes with indel events, we presented the length observed in two of the three specimens. b For trnM (cat), the value presented correspond to the average size among the three mitogenomes.
Specimens of the known species on the genus Metopiellus were collected in litter of ants or in caves (Asenjo, Ferreira & Zampaulo Rde, 2017). Unlike the other species of the genus, which have been recorded in forested areas or inside caves, the new species has been found in forested areas of the Serra dos Carajás, as well as in a savanna-like environment, although being less abundant in the latter.
For the first time, the mitochondrial genome of a Metopiellus species is described focusing on the phylogenetic position of Metopiellus crypticus sp. nov. within the subfamily Pselaphinae. In the obtained topology, the new species was recovered as sister to Batrisodes sp., although with low statistical support (BS = 42). However, this grouping was probably an artefact influenced by the absence of published mitogenomes of the others representatives of the other Metopiasini.
Despite of all three assembled mitogenomes presenting the standard structure sequence and gene content for Metazoa, we could not obtain a circularized assembly for any of them, probably due to a high repetitive DNA content in the D-loop control region (Sayadi et al., 2017), as indicated by the several mononucleotide repeats in both ends of the assembled sequences. Such a pattern has been frequently reported for beetle species, with several Coleoptera mitogenomes available in the GenBank database containing all expected genes, but missing part of the control region, and thus being reported as partial sequences.

Author Contributions
• Angélico Asenjo conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.
• Marcus Paulo Alves de Oliveira conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft.
• Renato R.M. Oliveira performed the experiments, analyzed the data, authored or reviewed drafts of the article, and approved the final draft.
• Eder Soares Pires performed the experiments, authored or reviewed drafts of the article, and approved the final draft.
• Marcely Valois analyzed the data, authored or reviewed drafts of the article, and approved the final draft.
• Guilherme Oliveira conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft.
• Santelmo Vasconcelos conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.

Field Study Permissions
The following information was supplied relating to field study approvals (i.e., approving body and any reference numbers): The samples were collected under the sampling permit 49.994, as granted by ICMBio/MMA.

DNA Deposition
The following information was supplied regarding the deposition of DNA sequences: The assembled mitogenomes are available at GenBank: MZ576843, MZ576844 and MZ576845.

Data Availability
The following information was supplied regarding data availability: